The results thus demonstrated that two distinct populations of limbal cells with SC characteristics can be isolated in the mouse. Both populations contained cells expressing the SP phenotype based on the efflux of Hoechst 33342 dye. However, forward-scattering analysis of SP cells from the top and bottom fractions showed that both populations differ in their size and granularity. The number of SP cells in the unseparated mouse limbus was 3.8% (average from five experiments) of total limbal cells, substantially higher than the number of slow-cycling corneal epithelial cells found at the mouse limbus
32 or the number of SP cells in human, rabbit, and rat limbal epithelia,
13 23 24 but corresponds to the number of SP cells found in the rat cornea.
23 Although it has been shown that the SP phenotype is associated with ABCG2 expression,
33 the corollary is not necessarily true (i.e., not all cells expressing ABCG2 exhibit the SP phenotype). The studies of Umemoto and coworkers in humans,
16 rabbits,
29 and rats
23 showed that although the number of cells exhibiting the SP phenotype was less than 2% in the limbus, immunochemistry revealed that a larger proportion (approximately 10%) of limbal basal epithelial cells expressed ABCG2 transporter.
23 Similarly, Budak et al.
18 suggested the existence of a significantly higher number of ABCG2
+ cells than SP cells. This discrepancy was explained by the differences in the transport activity of ABCG2. Umemoto et al.
23 also showed that in the rat, unlike the human and rabbit, the central cornea contains cells with the SP phenotype but that these cells expressed significantly lower levels of putative SC markers than did the SP cells in the limbus. In addition, SP cells found in the rat cornea had a different profile on forward scatter analyses than SP cells in the limbus. Our study showed that mouse limbal cells with the SP phenotype from the light cell fraction of the Percoll gradient had distinctive light-scattering properties from SP cells from the dense cell fraction. It appears that the light cell fraction resembles the SP cells described by Umemoto et al.
23 in the rat central cornea rather than the basal limbal SCs. A high expression of the corneal epithelial cell differentiation marker K12 in the light cell population supports this analysis. It is therefore apparent that interspecies differences exist in the distribution and properties of corneal epithelial cells with limbal SC characteristics and that the mouse may, in this respect, represent a unique species different from human, rabbit, or rat.